Multiresistant Pseudomonas aeruginosa
Overview Microbes are becoming more and more resistant to the drugs we use to treat their infections, and it is causing significant concern in hospitals around the world. A multiresistant strain of Pseudomonas aeruginosa of serotype O:1 was isolated from the sputum of a 52-year old male patient admitted to the ICU of the Teaching Hospital of Besancon, France. The strain is identified as PA2345, and displays notable drug resistance to aminoglycosides and various beta-lactams including third-generation cephalosporins and carbapenem. Drug resistance can occur in bacteria through gene transfer of resistance genes or through spontaneous mutations that activate intrinsic mechanisms of resistance such as efflux pumps, receptor alteration, and beta-lactamase formation. A genetic analysis was done to identify the specific mechanisms of resistance in PA2345. Genetic Analysis Production of ESBL (extended-spectrum beta-lactamse) was first noted in PA2345 with an antibiogram test, and confirmed using blaPER-1 specific PCR in accordance with two Pseudomonas strains isolated in the past decade. Strain RNL-1 serotype O:2 isolate in France in 1991, and PABG serotype O:12 isolate in Belgium in 1998. The PCR yielded one 925bp amplicon that had the indentical sequence to those of PABG and RNL-1. It is important to note however, that PA2345 remained confined to one patient and did not cause a hospital outbreak as PABG and RNL-1 did. This strain was revealed to not contain a plasmid, not transfer ceftazidime resistance by conjugation, and southern blot experiments revealed that the gene encoding for PER-1 production was located on the bacterial chromosome, which is consistent with PABG and RNL-1. Transposon Location Two 6-bp direct repeats on either side of 16-bp terminal inverted repeats that border insertion sequences are indicative of transposon insertion, and represent genetic organisation with composite transposons. The terminal inverted repeats are the binding sites for transposase enzymes. Interestingly the two insertion sequences, which have been called ISPa23 and ISPa24, showed limited amino-acid homology at 57%, which is notable dissimilarity between two transposase genes. Several composite transposons are known to encode for drug resistance, and this new composite transposon was called Tn2345. Amplification of PA2345, PABG, and RNL-1 with primers targeting the insertion sequences generated PCR products of identical lengths indicating the same if not very similar structures. Class I Integron In order to characterize the genetic determinents of the high level of PA2345 resistance to aminoglycosides a 5.8-kbSacI-SacI genetic fragment was cloned from PA2345 into E. coli DH5-alpha. The recombinant plasmid pAD1 causes resistance to kanamycin, gentamycin, and tobramycin, but not to amikacin and netilimicin. Sequencing of the pAD1 plasmid revealed the presence of a class I integron exhibiting a 5'-conserved segment (5' CS) carrying the integrase gene. Downstream, prior to the 3'CS of the class I integron were three gene cassettes: aaadB, orfE, and aadA11. The first gene cassette aaadB codes for aminoglycoside adenylyltransferase, which conferrs high resistance to kanamycin, gentamycin, and tobramycin. This strain has been reported in a Pseudomonas strain as a cassette carried by Class I integron, however in PA2345 no bla gene was found in the vicinity of aaadB. Downstream of aaadB is a orfE-like gene; it is 90% homologous to other sequenced orf genes, and encodes for aadA11, which is a gene that codes for streptomycin and streptinomycin resistance. Aminoglycoside Efflux in PA2345 Products of the aaadB and aadA11 genes do not confer resistance to amikacin, and don't account for the increased susceptibility of PA2345 to this specific aminoglycosidic agent. Stable overproduction of the MexXY-OrpM efflux pump system can increase susceptibility of P. aeruginosa to aminoglycosides including amikacin. So levels of mexX expression were measured with real-time PCR analysis. The results showed that PA2345 produced 34-times more mexX PAO1, the wild type, and 2-times more than MutGR1, a MexXY-overproducing mutant of PAO1, which is altered in the repressor gene mexZ. Sequencing of the mexZ of PA2345 only revealed silent mutations indicating that PA2345 is a mutant defective of an unknown regulation gene. Conclusion The analysis of multiresistant P. aeruginosa indicates that the acquired resistance arises from a variety of sources. Either by sequential exchanges of genetic material with transposons or integrons, or genetic mutations that initiate the altered expression of mechanism such as efflux pumps and target receptors that cause increased resistance. Geographically and genetically diverse strains of P. aeruginosa have displayed acquisition of the blaPER-1 gene, encoding for ESBL, suggesting that an unkown bacterial resevoir may be contributing to increasing occurences of multiresistant bacteria in Europe. This analysis identified the various mechanisms by which PA2345 has gained its specific resistances and susceptibilities including the class I integron, transposons, and gene cassettes. References [http://www.ncbi.nlm.nih.gov/pubmed/16451415 C. Lanes, C. Neuwrith, (2005): Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346794/ V. Aloush. S. Navon-Venezia (2006): MR Pseudomonas aeruginosa: Risk Factors and Clinical Impact] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346794/ ☁]